Role of cdc21+ and related genes in eukaryotic chromosome replication

Thesis



The Schizosaccharomyces pombe cdc21⁺ gene product is related to the Mcm2-3-5 family of replication proteins.

By phylogeny analysis of their protein sequences and screening for cdc21⁺-related sequences using molecular probes I have suggested that at least six types of cdc21⁺-related genes may be present in the yeast genome.

The isolation of interaction suppressors of the cdc21^ts mutant was attempted by overexpression of an S. pombe cDNA library. Two cDNAs were isolated, ts11⁺ and dom1⁺, whose overexpression specifically affected the viability of cdc21^ts cells under certain conditions. The predicted dom1 protein is 60% identical to the budding yeast HMG-like Nhp2 protein.

I have studied the phenotype of S. pombe cells overexpressing the cdc21⁺ gene and amino-terminal truncations of it. Overexpression of the cdc21⁺ gene caused cell elongation but cells were not significantly affected in growth rate. Cells overexpressing the carboxyl-terminal part of cdc21⁺ arrested in S phase and also entered mitosis in the absence of nuclear division.

The possibility that chromosomes in cdc21^ts arrested cells may be damaged was investigated by pulsed field gel electrophoresis. No differences could be found compared to wild-type chromosomes. I have also studied the arrest phenotype of cdc21 rad1 and cdc21 cdc2.3w double mutants. Both strains entered mitosis at the restrictive temperature indicating that cdc21^ts cells arrest in S phase and may contain DNA damage.

I have generated two new mutant alleles of cdc21⁺. The first allele was made by deleting most of the cdc21⁺ open reading frame (ORF). The second allele was constructed by placing the cdc21⁺ ORF under control a regulatable promoter. The resulting construct was used to complement the cdc21 deletion. Both mutants were inviable under appropriate conditions arresting in S phase as elongated cells, although a proportion of them (15-20%) entered mitosis in the absence of nuclear division.

Authors: Maiorano, D; Maiorano, Domenico

• Publication date: 1995
• DOI: 10.5287/ora-nb1przbv5
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Genetics; Cytology; Schizosaccharomyces pombe